Method for producing cucumber pachytene chromosomes
A chromosome and pachytene stage technology, applied in the field of plant biology, can solve the problems of low utilization rate of cucumber anthers, unclear chromosome morphology, etc., and achieve the effects of reducing the time for allocating anthers, saving cucumber seeds, and improving the utilization rate
Inactive Publication Date: 2013-05-22
NANJING AGRICULTURAL UNIVERSITY
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[0005] The purpose of the method for producing cucumber pachytene chromosomes of the present invention is to solve the defects of severe chromosome entanglement, unclear chromosome shape, etc., and low utilization rate of cucumber anthers during the production of cucumber pachytene chromosomes in the past, and to invent a method that can prepare high dispersion. Cucumber pachytene chromosomes, and increased the utilization of cucumber anthers, laid the foundation for the production of high-precision physical maps of cucumber chromosomes, and accelerated the progress of cucumber cytology research
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[0025] The implementation procedure of chromosome preparation at the thick line stage of cucumber of the present invention includes:
[0026] (1) Material preparation: Take appropriate size flower buds at the early and full blooming stage of cucumber plants, and place them in a 3:1 E:G (ethanol: glacial acetic acid) fixative for at least 24 hours. Change the flower buds to 70% ethanol and set aside.
[0027] (2) Select the anthers in the thick line stage: take out a fixed flower bud, separate the anthers under a microscope, take a quarter of the anthers on a clean glass slide, and add a few drops of 45% acetic acid. Soften, tap the anthers gently with tweezers, cover with a cover glass, press gently, and then observe under an ordinary optical phase contrast microscope. If the chromosomes are observed to be in the thick line stage, place the remaining anthers Put it into a 1.5ml centrifuge tube, add a small amount of E:G fixative, and store at 4°C for later use.
[0028] (3) Enzymat...
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The invention discloses a method for producing cucumber meiosis pachytene chromosomes. The method comprises the following steps: taking a flower bud of a cucumber full-bloom stage plant, fixing the flower bud in a fixing solution containing ethanol and glacial acetic acid with the volume ratio of ethanol to glacial acetic acid (E:G) of 3:1, stripping off the flower bud, performing enzymolysis on the anther in meiosis pachytene by using cellulase and pectinase, mashing the anther subjected to enzymolysis in a proper amount of E:G fixing solution, sucking the suspension, smearing, and producing the pachytene chromosomes by using a flame drying method. By adoption of the smearing and flame drying method, the pachytene chromosomes which are high in dispersion degree and good in shape can be produced, and the utilization rate of cucumber anther is improved; and more important, the obtained pachytene chromosomes can be directly used for fluorescence in-situ hybridization, and a reliable guarantee is provided for cucumber cytology research.
Description
1. Technical Field [0001] The invention discloses a method for preparing chromosomes at the thick line stage of cucumber, which is specially used for preparing the thick line stage chromosomes of cucumber, is beneficial to carry out cytological research of cucumber, and belongs to the field of plant biotechnology. 2. Technical background [0002] Cucumber (Cucumis sativus L., 2n=14) is an annual trailing herbaceous cucumber in the Cucumber family of the Cucumber family. It originated in the tropical rain forest area of the southern foothills of the Himalayas. It has been cultivated in my country for more than 2,000 years. Cucumber is convenient to eat, rich in vitamin C and a variety of beneficial minerals. It is one of the main vegetable crops in my country, and it has played a huge role in improving people's living standards and meeting the annual supply of vegetables. my country is the largest cucumber producer and consumer in the world. According to statistics, the cultivat...
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Inventor 娄群峰何玉华陈劲枫贾利李季
Owner NANJING AGRICULTURAL UNIVERSITY